MACROMOLECULAR BINDING OF ALDOSTERONE IN TOAD BLADDER

Protein-bound aldosterone has been extracted by sonication of nuclei from the mucosal cells of toad bladder. After incubation of hemibladders in 10 and 100 nM (+)-[3H]aldosterone, 1.5·10-14 and 7.7·10-14 moles bound aldosterone per 100 μg DNA can be extracted, respectively. Most of the bound aldosterone dissociates rapidly, and 4 h after isolation only a stable component of 20% remains. Dissociation is accelerated at room temperature. The stability of the complex is unaffected by dithiothreitol but enhanced by 25% glycerol. An excess of deoxycorticosterone, hydrocortisone, spirolactone (SC 14266) and unlabeled aldosterone, when added to the incubation medium, displace bound aldosterone while testosterone has no effect on binding by purified nuclei. This suggests mineralocorticoid specificity for the binding protein. [3H]Hydrocortisone (100 nM), a less active mineralocorticoid than aldosterone, binds primarily to the nonspecific, nondissociable binding sites at this concentration. One high-molecular-weight component and one component of molecular weight of approx. 100 000 were demonstrated by agarose column chromatography. Both components are stable and unaffected by deoxycorticosterone. The mineralocorticoid-specific complex can be equated with the rapidly dissociable fraction. When nuclei are sonicated and (+)-[3H]aldosterone is then added a nonsaturable nonspecific binding occurs which is increased with time and unaffected by temperature. Thus three aldosterone binding complexes have been isolated from toad-bladder mucosal-cell nuclei one of which is rapidly dissociable and mineralocorticoid specific. © 1969.